ANTIMICROBIAL ACTIVITY OF SUPERCRITICAL CARBON DIOXIDE EXTRACTS OF TUBEROSE (POLIANTHES TUBEROSA LINN.) FLOWERS AGAINST COMMON PATHOGENS

Received on 28 October, 2013; received in revised form, 04 January, 2014; accepted, 09 March, 2014; published 01 April, 2014

ANTIMICROBIAL ACTIVITY OF SUPERCRITICAL CARBON DIOXIDE EXTRACTS OF TUBEROSE (POLIANTHES TUBEROSA LINN.) FLOWERS AGAINST COMMON PATHOGENS

Probir Kumar Ghosh ¹, Paramita Bhattacharjee*¹ and Satadal Das ²

Department of Food Technology and Biochemical Engineering, Jadavpur University ¹, Kolkata 700032, West Bengal, India

Department of Microbiology, Peerless Hospital & B. K. Roy Research Center ², Kolkata 700094, West Bengal, India

ABSTRACT: Tuberose flower extracts were obtained by green technology of Supercritical carbon dioxide (SC-CO₂) extraction and solvent extractions (using n-hexane and petroleum ether of b.p. 60-80 °C). Antimicrobial activities of these extracts were tested in vitro against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Helicobacter pylori, Vibrio cholerae and Candida albicans. No activity was found against H. pylori and V. cholerae. Antimicrobial activities of the extracts were established by minimum inhibitory concentration (MIC) assays against the remaining test microorganisms. The SC-CO₂ and solvent extracts showed potency against all test microorganisms. SC-CO₂ extract obtained at 40 °C, 100 bar pressure, 135 min extraction time, and n-hexane extract showed most promising results, among all the extracts. However, considering the global need for green extracts for therapeutic applications, SC-CO₂ extraction is the recommended technique for extracting antimicrobials from tuberose flowers. The chemical constituents conferring antimicrobial potencies to the extracts were also identified by GC/MS analyses.

Tuberose, Solvent extraction, Supercritical carbon dioxide extraction, Antimicrobial activity, Minimum inhibitory concentration

INTRODUCTION:Tuberose (Polianthes tuberosa Linn.) is a herb and a member of the Agavaceae family. It is commonly known as ‘Rajnigandha’ in India. Bioactive constituents are extracted from herbs conventionally in aqueous and organic solvents which leave artifacts and solvent residues in the extracts, posing health and environmental concerns. These problems have been averted by use of supercritical fluid (SCF) extraction technology.

This is a green technology that uses fluids of GRAS status in the supercritical state as extracting solvents. CO₂ is the most commonly used SCF because of its non-toxicity, non-flammability and low critical temperature. Supercritical carbon dioxide (SC-CO₂) extractionhas gained paramount importance in extraction of bioactive constituents from several natural matrices, including flowers, such as chrysanthemum ¹ and jambú ².

The present study envisages SC-CO₂ extractionof bioactive constituents from tuberose flowers for possible usage as antimicrobial agents against common pathogenic microorganisms. Reports on antimicrobial activity of tuberose flower extracts against bacteria (by zone of inhibition) ³ and fungi (reduction in % mycelia growth) ⁴ are scanty; moreover, none have reported on MIC (minimum inhibitory concentration) values of the extracts. MIC is regarded as a ‘gold standard’ to determine susceptibility of microorganisms to antimicrobials ⁵. In the present investigation, the extracts obtained by SC-CO₂ have been assessed for antimicrobial efficacies against pathogenic bacteria and fungi, by determining their MIC values, and the compounds attesting to their antimicrobial potencies have been identified by GC/MS. Conventional solvent extraction has also been conducted in this study as an experimental control.

To the best of our knowledge, this paper reports for the first time on MIC values of tuberose extracts. Moreover, there is no literature on SC-CO₂ extraction of antimicrobials from tuberose flowers.

MATERIALS AND METHODS:

Materials:Tuberose flowers (Polianthes tuberosa Linn.) of Calcutta single variety were procured from cultivators of Barasat, 24 Parganas North, West Bengal (22° 71’ N and 88° 51’ E, at about 13 m elevation above mean sea level, located in the eastern Gangetic plain of India) in February 2013 and were authenticated by West Bengal Food Processing and Horticulture Development Corporation Limited, Kolkata, India. The flowers were cultivated under tropical-temperate climate (24-32°C, 75-85% RH) in sandy loamy soil of pH 7-8. The flowers were air dried in shade at room temperature (23 ± 2°C) to constant weight. The moisture content of fresh flowers was estimated to be 67.66 % on a dry weight basis, by Bidwell-Sterling method (AOAC 1990; method 925.04) ⁶.

Petroleum ether (b.p. 60-80°C), n-hexane and ethanol (96 %) were procured from M/s Merck Ltd., India. Muller-Hinton agar and broth (MHA and MHB, respectively), Brain Heart Infusion (BHI) agar (M/s Difco Laboratories, MI, USA), horse serum (M/s Invitrogen, NY, USA), IsoVitaleX (M/s Becton-Dickson, MD, USA), Vancomycin, Polymixin B and Trimethoprim (M/s Sigma, MO, USA) were used for microbial culture. CO₂ (food grade) was procured from M/s BOC India Ltd., Kolkata, India. All reagents were of AR grade.

FIGURE 1: SCHEMATIC DIAGRAM OF SC-CO₂ EXTRACTION SYSTEM USED IN THIS INVESTIGATION

Extraction of tuberose extracts using SC-CO₂ and solvent extraction:

SC-CO₂ extraction of tuberose flowers:For SC-CO₂ extraction, a SPE-ED SFE 2 model of M/s Applied Separations, PA, USA was used. The schematic diagram of a SC-CO₂ extraction unit of SPE-ED SFE 2 model employed in this study, is shown in Fig. 1. Three different unit operations are involved in this system such as extraction, expansion and separation and their corresponding critical components are high-pressure extractor, a pressure reduction valve and a low pressure extractor, respectively. This system can be performed in either static or in dynamic mode.

The operational limit of the system is 240°C and 690 bar. The sample is charged into a 50 ml extraction vessel (SS 316). The sample bed is firmly packed to ensure that the supercritical fluid diffuses uniformly through the sample matrix. The voidage is filled with Spe-ed Matrix (silica matrix). Polypropylene frits are placed on both ends of the sample bed with tight end-fittings.

The extraction vessel is kept in the oven module and the Z-type thermocouple is connected to the vessel body which detects the vessel temperature. The desired temperature for SC-CO₂ extraction is achieved through regulatory switches of oven module. The food grade CO₂ from the cylinder is fed to the extractor through a high pressure modifier pump (Speed MAX P/N 7025) fitted with refrigerated cooling bath to chill the ‘pump module’ head at -2°C.

CO₂ is passed through the extraction vessel for a definite period of time during which the outlet valve is kept closed. The first mode of extraction is known as static conditioning to allow sufficient contact between sample matrix and extractant. During the dynamic phase of extraction, the outlet valve is kept open, the soluble analytes are portioned from the bulk of the sample matrix into the SC-CO₂, and the extract laden CO₂ is swept through a flow restrictor into a separator or collection vial, kept at ambient conditions. A bubble flow meter attached at the collection end of the extraction module indicates the flow rate of gaseous CO₂. The desired flow rate is achieved through a micrometering valve present in the collection module. At reduced temperature and pressure conditions, the extract precipitates with reduction of fluid density in the separator (collection vial) and CO₂ in gaseous form vents out into the atmosphere.

The batch size, extraction time and flow rate of CO₂ were fixed by preliminary experimental trials. SC-CO₂ extraction was conducted with 20 g dry flowers at extraction conditions of 40°C, 100 bar; 40°C, 200 bar; 60°C, 100 bar; 60°C, 200 bar; 80°C, 200 bar and 80°C, 300 bar; keeping extraction time of 135 min (120 min static time + 15 min dynamic time) constant, at a flow rate of 1 l/min of gaseous CO₂. Three independent extraction runs were conducted for a given set of extraction conditions, with three independent batches of dried tuberose flowers.

All extracts were collected into 96 % ethanol in screw capped glass vials, kept in an ice bath. Post collection, the extracts were purged with nitrogen to evaporate ethanol and the yields were determined gravimetrically. The extracts were then redissolved in 96 % ethanol  and stored in N₂ purged screw capped amber colored vials at -18 °C in dark, until further analyses.

Solvent extraction of tuberose flowers:Solvent extractions were carried out in Soxhlet extraction assembly using n-hexane and petroleum ether (b.p. 60-80°C), individually, for 8 h. Post extraction, the solvents were evaporated in Rotavac (M/s Eyela Corp., Japan) at 40-45°C, 50 mbar, and the yields were assayed gravimetrically. The extracts were subsequently dissolved in ethanol (96 %) and stored as above.

Antimicrobial activity assay of tuberose extracts:The test microorganisms used in this study included bacteria such as Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Helicobacter pylori (strain 26695) and Vibrio cholerae (strain O1) and a fungi Candida albicans (ATCC 10231). These microorganisms were selected since they are the common pathogens known to cause diseases of skin, GI tract and urinary tract infections, enteric ulcer, cholera and candidiasis.

Disc diffusion method was employed for screening antimicrobial potencies of the extracts using MHA for all the microorganisms, except for H. pylori. H. pylori was cultured on Brain Heart Infusion (BHI) agar supplemented with 5 % Horse serum, 0.4 % IsoVitaleX, Trimethoprim (5 µg/ml), Vancomycin (8 µg/ml) and Polymixin B (10 µg/ml). Autoclaved Whatman No. 1 filter paper discs (5 mm diameter) were each spiked with 10 µl of the extracts, and placed on the microorganism swabbed agar plates and incubated in a BOD incubator at 37°C for 24 h (except for H. pylori). The plate with H. pylori was incubated at 37°C in microaerophilic atmosphere (5 % O₂, 10 % CO₂, 85 % N₂) (double gas incubator; M/s Heraeus, Langenselbold, Germany) for 3 days.  Post incubation, extracts showing inhibition zones were subjected to assay of MIC. H. pylori and V. cholerae showed no inhibition zone for all the extracts and therefore, were not subjected to MIC assays.

For determination of MIC values, the inoculums of the microorganisms were prepared in MHB and adjusted to 0.5 McFarland turbidity standard (10⁶-10⁷ cfu/ml of microorganisms). MIC assays were conducted by the ‘broth microdilution method’ and the microtiter plates were incubated in a BOD incubator at 37°C for 24 h. A microplate reader (M/s Micronaut System, Germany) was used to measure the optical density of the microplate at 0 h and 24 h at 620 nm. To ascertain the static and cidal activities, the extracts showing MIC were transferred from the 96-well microplate on to MHA plates. Microbial growth after 24 h of incubation at 37°C was considered to indicate static and not cidal activity of the extracts against test microorganisms. For MIC assay, positive control used were Gentamycin for bacteria and Amphotericin B for fungi; while ethanol (96 %) used as a diluent for the extracts, was considered as the negative control.

GC/MS analyses of tuberose extracts:To identify the compounds responsible for antimicrobial activity in the tuberose extracts, GC/MS analysis was carried out in Perkin Elmer GC Clarus^® 680 chromatograph (M/s Perkin Elmer, MA, USA) with Elite-5 MS (Perkin Elmer, MA, USA) fused silica capillary column (30 m x 0.25 mm i.d.; 0.25 µm film thickness) coupled to a Perkin Elmer Clarus^® SQ 8T Mass Detector (M/s Perkin Elmer, MA, USA). The GC injector and MS transfer line temperatures were set to 230°C and 270°C, respectively. The oven temperature was programmed as follows: 50°C for 5 min, then an increase from 50 to 250°C at 5°C / min and finally a fixed isothermal hold at 250°C for 10 min. The carrier gas was He at a flow rate of 1 ml/ min. 1µl of extract was injected at a split ratio of 1:50. The ionization of the sample was performed in the EI ion source (70 eV) and the acquisition mass range was set at 35-500 amu. Identification of components was based on comparison of their mass spectra (using molecular ion [M⁺] peak, base peak and the m/z values) with those provided in mass spectra library NIST (2007) and in literature ^{7, 8}. The compounds whose mass spectra matched with references ^{9, 10} were only confirmed. The relative peak area percentages (without correction factor) were used to report the abundance of a compound in the extracts.

Statistical analyses:Statistical analyses were carried out by one-way ANOVA at 95 % confidence interval using STATISTICA 8.0 software (Statsoft, OK, USA) to analyze the effects on MIC values of the test microorganisms with the solvents.

RESULTS:

Preliminary screening of extracts by disc diffusion method:The disc diffusion method showed that SC-CO₂ extracts obtained at 40°C, 100 bar; 60°C, 200 bar and 80°C, 300 bar and with n-hexane and petroleum ether (b.p. 60-80°C) were potent against the test microorganisms. Therefore, gravimetric yield, color and appearance and GC/MS analyses of only these extracts have been reported.

TABLE 1: YIELD, COLOR AND APPEARANCE OF THE EXTRACTS OBTAINED BY SOLVENT AND SC-CO₂ EXTRACTION TECHNIQUES USED FOR MIC ASSAY

^a Centrifugation was done at 1000g, 20 min and supernatant was used for GC/MS analyses. ^b Yield of extracts are mean ± SD from three independent extractions.  ^{c, d, e, f, g} Different letters in a column indicate significant difference at p < 0.05.

Yield, color and appearance of extracts considered for MIC assay:Among the SC-CO₂ extracts, the highest yield of extract was obtained at 80 °C, 300 bar; and lowest at 40 °C, 100 bar (Table 1). We also observe that yield of extract obtained by n-hexane was lower than that obtained by petroleum ether.

All SC-CO₂ extracts were clear in appearance and light yellow in color, except that obtained at 80°C, 300 bar which was light green and turbid. The extract obtained using n-hexane was yellowish orange and slightly waxy, while a light orange colored waxy extract was obtained with petroleum ether.

Antimicrobial activity of Tuberose extracts by MIC assay:

Antimicrobial activity of extracts obtained by SC-CO₂:ANOVA study concluded that the SC-CO₂ extract obtained at 40°C, 100 bar showed significant difference (p = 0.00) in MIC values obtained for the different test microorganisms, with best activity against C. albicans (Table 2). The extract obtained at 60°C, 200 bar showed significant difference (p = 0.00) in MIC values and the best potency against P. aeruginosa. The extract obtained at 80°C, 300 bar also showed significant difference (p = 0.00) in MIC values with high antimicrobial efficacies against P. aeruginosa and E. coli (Table 2).

Antimicrobial activity of extracts obtained by solvent extraction:From ANOVA study (Table 2), we observed that the extracts obtained by n-hexane showed significant differences (p = 0.00) in the MIC values for the different test microorganisms. This extract showed best potency against P. aeruginosa. The extracts obtained by using petroleum ether also showed significant difference (p = 0.00) in the MIC values with respect to these microorganisms. This extract showed best antimicrobial activity against C. albicans;however, noactivity was found against S. aureus.

TABLE 2: IN VITRO MINIMUM INHIBITORY CONCENTRATION (MIC) VALUES OF TUBEROSE EXTRACTS^a AGAINST TEST MICROORGANISMS^b

^a Extracts were dissolved in ethanol for antimicrobial assays.

^b For H. pylori and V. cholerae MIC assay was not conducted since they did not show zone of inhibition by disc diffusion method.

^c MIC of extract higher than that of negative control and therefore not considered to have antimicrobial potency.

^d,e,f,g Different letters in a column indicate significant difference at p < 0.05.     

^m MIC values are mean ± SD of three independent experiments from three independent extracts.

ⁿ MIC value of Gentamycin (antibacterial positive control).

^p MIC value of Amphotericin B (antifungal positive control).

GC/MS analyses of tuberose extracts:

GC/MS analyses of SC-CO₂ extracts: Fig. 2a and Table 3 represent the chromatogram and compound profiles of the SC-CO₂ extract at 40 °C, 100 bar, obtained by GC/MS analysis. Methyl eugenol was found to be the most abundant compound in this extract. The chromatogram shown in Fig. 2b and the compound list in Table 4 describes the SC-CO₂ extract at 60°C, 200 bar. Styrene was found to be the most abundant compound among the compounds identified. From Fig. 2c and Table 5, for SC-CO₂ extract at 80°C, 300 bar, eugenol was found to be the most abundant compound identified. Overall, SC-CO₂ extract showed lesser proportion of hydrocarbons (hentriacontane) vis-à-vis the solvent extracts, demonstrating increased solubility and selectivity of SC-CO₂ for benzene compounds.

GC/MS analyses of solvent extracts:Fig. 3a represents the chromatogram of the extract obtained by n-hexane and Table 6 lists the peaks of identified compounds in the same. Among the benzene compounds, trans-carveol has been tentatively identified and found to be the most abundant. The chromatogram of compounds in petroleum ether (b.p. 60-80°C) extract has been shown in Fig. 3b and the identified compounds have been presented in Table 7. We found cis-3-methyl cyclopentanol to be the most abundant compound in this extract.

FIGURE 2: GC/MS CHROMATOGRAMS OF TUBEROSE EXTRACTS OBTAINED BY USING SC-CO₂ AT (a) 40°C AND 100 BAR, (b) 60°C, 200 BAR AND (c) 80°C, 300 BAR. LABELS ON THE CHROMATOGRAMS HAVE BEEN EXPLAINED IN TABLES 3, 4 AND 5, RESPECTIVELY.

TABLE 3: LIST OF COMPOUNDS IDENTIFIED BY GC/MS USING SC-CO₂ (40°C, 100 BAR)

R.T.: Retention time.

n.a.: not applicable. 

^a Identifications were carried out using NIST library (2007), Adams (2007), NIST mass

spec data centre (2013) and literature review.

^b,c,d,e,f Compounds having antimicrobial activity already reported in literature.

^g Area percentages were reported without correction factor.

m/z values of the identified compounds:

acetophenone: 39, 43, 51, 68, 74, 77, 78, 91, 105, 106, 120.

benzoic acid methyl ester: 38, 51, 77, 105, 106, 136.

eugenol: 43, 51, 55, 65, 77, 81, 91, 103, 122, 131, 137, 149, 163, 164.

alpha farnesene: 39, 41, 43, 55, 67, 69, 79, 93, 107, 119, 135, 147, 161, 189, 204.

beta bisabolene: 39, 41, 43, 55, 67, 69, 79, 91, 93, 109, 119, 135, 147, 161, 189, 204.

benzyl benzoate: 39, 51, 61, 65, 77, 91, 105, 106, 121, 152, 167, 194, 212.

TABLE 4: LIST OF COMPOUNDS IDENTIFIED BY GC/MS USING SC-CO₂ (60°C, 200 BAR)

R.T.: Retention time.

n.a.: not applicable.

   ^a Identifications were carried out using NIST library (2007), Adams (2007), NIST mass spec

data centre (2013) and literature review.

^b,c Compounds having antimicrobial activity already reported in literature.

^d Area percentages were reported without correction factor.

m/z values of the identified compounds:

styrene: 39, 51, 63, 78, 103, 104.

benzaldehyde: 39, 51, 63, 77, 78, 105, 106.

1,3 dicloro benzene: 51, 55, 74, 75, 111, 113, 146.

1-phenyl pentanone: 41, 51, 53, 77, 78, 105, 106, 120, 162.

1-chloro octadecane: 43, 55, 57, 71, 85, 91, 105, 111, 133, 147, 162.

8-heptadecene: 41, 43, 55, 57, 67, 69, 83, 97, 111, 125, 130, 156, 168, 207, 219, 238.

TABLE 5: LIST OF COMPOUNDS IDENTIFIED BY GC/MS USING SC-CO₂ (80°C, 300 BAR)

R.T.: Retention time.

n.a.: not applicable.

^a Identifications were carried out using NIST library (2007), Adams (2007), NIST mass spec

data centre (2013) and literature review.

^b,c Compounds having antimicrobial activity already reported in literature.

^d Area percentages were reported without correction factor.

m/z values of the identified compounds:

4-(2-propenyl) phenol: 39, 51, 55, 65, 77, 79, 91, 105, 107, 117, 131, 133, 134.

FIGURE 3: GC/MS CHROMATOGRAMS OF TUBEROSE EXTRACTS OBTAINED BY USING SOLVENT EXTRACTION USING (a) n-HEXANE (b) PETROLEUM ETHER (B.P. 60-80°C). LABELS ON THE CHROMATOGRAMS HAVE BEEN EXPLAINED IN TABLES 6 AND 7, RESPECTIVELY.

TABLE 6: LIST OF COMPOUNDS IDENTIFIED BY GC/MS USING n-HEXANE AS SOLVENT

R.T.: Retention time.

n.a.: not applicable.

^a Identifications were carried out using NIST (2007), Adams (2007) NIST mass spec data

centre (2013) and literature review.

^b,c,d Compounds having antimicrobial activity already reported in literature.

^e Area percentages were reported without correction factor.

^f Tentative identification was possible since the mass spectra of the compound could not be

matched with available standard mass spectra libraries.

m/z values of identified compounds:

p-xylene: 39, 51, 65, 77, 91, 92, 105, 106.

1-ethyl 3-methyl benzene: 39, 51, 65, 77, 91, 105, 120.

mesitylene: 39, 51, 65, 77, 91, 105, 106, 119, 120.

limonene: 41, 53, 65, 67, 68, 79, 80, 91, 92, 93, 94, 107, 121, 136.

1,4 diethyl benzene: 41, 51, 65, 77, 91, 105, 119, 134.

methyl eugenol: 41, 51, 65, 77, 91, 107, 115, 135, 147, 163, 178.

2,4 bis (1,1 dimethyl ethyl) phenol: 41, 57, 74, 91, 107, 115, 147, 163, 175, 191, 206.

pentacosane: 43, 57, 71, 85, 99, 113, 127, 155, 169, 211, 225, 253, 267, 352.

hentriacontane: 45, 55, 57, 69, 71, 85, 99, 113, 127, 155, 169, 197, 211, 239, 253, 281, 436.

TABLE 7: LIST OF COMPOUNDS IDENTIFIED BY GC/MS USING PETROLEUM ETHER, B.P. 60- 80°CAS SOLVENT

R.T.: Retention time.

n.a.: not applicable.

^a Identifications were carried out using NIST library (2007), Adams (2007), NIST mass

spec data centre (2013) and literature review.

^b,c,d,e Compounds having antimicrobial activity already reported in literature.

^f Area percentages were reported without correction factor.

m/z values of the identified compounds:

cis-3-methyl cyclopentanol: 41, 56, 57, 68, 71, 82, 100.

1,2,4 trimethyl benzene: 39, 51, 65, 77, 79, 91, 105, 119, 120.

salicyl hydrazide: 39, 53, 65, 76, 93, 121, 122, 151.

1,6 dimethyl napthalene: 49, 69, 85, 98, 115, 141, 156, 157.

benzophenone: 39, 51, 76, 77, 78, 105, 106, 152, 181, 182.

DISCUSSION: For SC-CO₂ extracts, highest gravimetric yield (significant at p = 0.00) was obtained at extraction conditions of 80°C, 300 bar, followed by those at 60°C, 200 bar and 40°C, 100 bar. The highest yield at 80°C, 300 bar may be attributed to extraction of oleoresins which are known to be extracted from plant materials at a pressure regime of 250-350 bar ¹¹. Moreover, these oleoresins are viscous in nature, which could have rendered the extract waxy and turbid; unlike the extracts obtained at lower pressure regimes.  ANOVA study on the yield of the solvent extracts indicated that the gravimetric yield of the petroleum ether extract was significantly higher (p = 0.00) than that of the n-hexane extract.

From the overall antimicrobial activities of SC-CO₂ extracts, we found best potency of the extract obtained at 40°C, 100 bar against C. albicans. All the SC-CO₂ extracts were equipotent against P. aeruginosa. The extract obtained at 80°C, 300 bar was most potent against E. coli (Table 2). The significant differences in MIC values of the extracts can be attributed to extraction of selective compounds by SC-CO₂ at different temperature-pressure regimes which could be owing to differences in the solubilities of these compounds in the extracting solvent under different extracting conditions. The SC-CO₂ extract at 40°C, 100 bar was found to have maximum potency against all the test microorganisms (Table 2). We found that tuberose extracts obtained by SC-CO₂ and solvents, have bacteriostatic and fungistatic activities. This is in agreement to the fact that MIC represents the bacteriostatic and not bactericidal concentration of an antimicrobial agent ¹². Further, GC/MS analyses of the extracts identified compounds attesting to antimicrobial potencies.

In the SC-CO₂ extract at 40°C, 100 bar (Table 3), compounds with antimicrobial potency such as acetophenone ¹³, alpha-farnesene and beta-bisabolene ¹⁴, eugenol ¹⁵ and methyl eugenol ¹⁶ have been identified. For the extract obtained at 60°C, 200 bar, in addition to eugenol, styrene was also detected which is known to be an antimicrobial agent ¹⁷, shown in Table 4. In the extract obtained at 80°C, 300 bar, the presence of eugenol and methyl eugenol attest to antimicrobial activities (Table 5).

Furthermore, in the SC-CO₂ extract at 40 °C, 100 bar, compounds such as beta bisabolene and alpha farnesene have been identified. We hypothesize that these compounds synergistically contribute to the overall antimicrobial potency of the extract which could be selectively extracted under these extracting conditions. These compounds have not been identified in the extracts obtained at 60°C, 200 bar and at 80°C, 300 bar; which establishes selective extraction by SC-CO₂ at 40°C, 100 bar. It can thus be concluded that the extract obtained at 40°C, 100 bar has the best antimicrobial activity (Table 2).

GC/MS analyses of n-hexane and petroleum ether extracts (Tables 6 and 7) confirmed the presence of compounds with antimicrobial potencies such as p-xylene ¹⁸, limonene ¹⁵ and methyl eugenol. Additionally, salicyl hydrazide, a potent antimicrobial ¹⁹ was also identified in the petroleum ether extract (Table 7).

CONCLUSIONS: MIC assays of SC-CO₂ and solvent extracts of tuberose flowers attested tuberose to be a potential natural source of antimicrobials against S. aureus, P. aeruginosa, E. coli and C. albicans; but not against H. pylori and V. cholerae. The SC-CO₂ extract obtained at 40 °C, 100 bar pressure, 135 min extraction time, and that by n-hexane extract showed most promising results as antimicrobials against all test organisms. The chemical compositions of the extracts have been ascertained by GC/MS analysis confirming chemical identities of constituents possessing antimicrobial potencies. Owing to international regulatory concerns, the green SC-CO₂ extract alone is advocated for therapeutic applications. We recommend topical application studies using this extract, since some of its constituents are known to be toxic for internal usage.

ACKNOWLEDGEMENT: The authors would like to thank Ministry of Food Processing of India (MoFPI), New Delhi, India, for providing financial assistance for the study. We are also thankful to Dr. Ashish Kumar Mukhopadhyay, National Institute of Cholera and Enteric Diseases (NICED), Kolkata; Dr. Tapan Kanti Pyne and Sayantan Paria (SRF),  Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Kolkata and Prof. Somnath Ghosh, Dept. of Chemistry, Jadavpur University, Kolkata.

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    How to cite this article:

    Ghosh PK, Bhattacharjee P and Das S: antimicrobial Activity of supercritical carbon dioxide extracts of tuberose (Polianthes tuberosa Linn.) flowers against common pathogens. Int J Pharm Sci Res 2014; 5(4): 1279-89.doi: 10.13040/IJPSR.0975-8232.5(4).1279-89

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